Mycobacterium kansasii is a faculative pathogen which can cause a severe tuberculosis-like disease in man. Approximately 5% of mycobacterial isolates from AIDS patients have been identified as M. kansasii. Because of the clinical importance of this nontuberculous mycobacterial species, we have prepared a M. kansasii lgt11 gene expression library. To screen the gene library, we have generated 6 monoclonal antibodies directed against M. kansasii antigens. Three of these antibodies react specifically with M. kansasii sonic extracts. Previously, we have identified a clone from the library which expressed a recombinant antigen comprised of the 35 kDa antigen from M. kansasii (MK35). This protein was shown to be immunoreactive in serological and skin test assays. To determine the skin test specificity of MK35, the recombinant antigen was purified and injected intradermally into sensitized guinea pigs. This antigen elicited a large delayed-type hypersensitivity response in M. kansasii sensitized pigs, a moderate response in animals sensitized with M. avium, and no reaction in guinea pigs immunized with M. tuberculosis. Because of the immunoreactivity and skin test specificity of MK35, DNA sequencing of the gene encoding MK35 has been initiated. Thus far, approximately 75% of the gene sequence has been determined.